Mature microRNAs1 (miRNAs) are endogenous, ˜21 nucleotide (nt), non-coding RNAs whose primary function is believed to be translational repression of protein coding mRNAs. The mature miRNA is processed from longer precursor molecules by the enzymes drosha and Dicer. 1 The abbreviations used are: cDNA, complementary DNA; C. elegans, Caenorhabditis elegans; CLL, chronic lymphocytic leukemia; LMW, low molecular weight; miRNA, microRNA; mRNA, messenger RNA; nt, nucleotides; PCR, polymerase chain reaction; pri-miRNA, primary microRNA precursor; RT-PCR, reverse transcriptase polymerase chain reaction; Tm, melting temperature.
miRNAs have been found in C. elegans, Drosophila, plants, mice and humans, suggesting an ancient and widespread role for these non-coding RNAs. To date, over 3,500 miRNAs have been discovered, including 114 in C. elegans, 332 in humans and 270 in mice. An algorithm termed miRscan was developed to predict the number of miRNAs in a genome based upon the phylogenetically conserved foldback structure of the miRNA. miRscan predicts that the total number of miRNAs in the human genome to be 200-255, or about 1% of the predicted genes in humans.
The founding members of the miRNA class of genes, lin-4 and let-7, are expressed temporally during development of C. elegans. In addition to regulating development in C. elegans, miRNAs have been shown to negatively regulate the proapoptotic gene hid during Drosophila development. Thus, levels of miRNA or miRNA precursors in samples taken from C. elegans or Drosophila can be used to determine the stage of development of these two organisms.
miRNAs are also associated with various diseases. For example, two human miRNAs (miR-15a and miR-16) have been mapped to the region 13q14 that is commonly deleted in chronic lymphocytic leukemia (CLL). The expression of miR-15a and miR-16 is reduced in CLL patients with loss of heterozygosity at 13q14.
Thus, the levels of miRNA and their precursors in samples taken from a test subject, including human subjects, can be used to study the role of miRNAs in health and disease and to identify drugs that modulate miRNA function.
Most of the miRNA expression data published to date have used Northern blotting to detect both the mature and pre-miRNA precursors. Probes designed to hybridize to the mature miRNA detect the ˜22 nt mature miRNA and the ˜75 nt pre-miRNA simultaneously on the blot. Primer extension has also been effectively used to detect the mature miRNA. As tools for monitoring gene expression, gel based assays (Northern blotting, primer extension, RNase protection assays, etc.) have disadvantages, including low throughput and poor sensitivity.
cDNA microarrays are an alternative to Northern blotting to quantify miRNAs since microarrays have excellent throughput. For example, a recent report used cDNA microarrays to monitor the expression of miRNAs during neuronal development. Microarrays have other disadvantages including the necessity for high concentrations of input target for efficient hybridization and signal generation, poor sensitivity for rare targets and the necessity for post-array validation using more sensitive assays such as real-time PCR.
A PCR approach has been used to determine the expression levels of mature miRNAs. This method, while useful to clone miRNAs, is impractical for routine gene expression studies since it involves gel isolation of small RNAs and ligation to linker oligonucleotides. PCR has also been used to measure the expression of primary miRNA precursor molecules.
Because of the short size of miRNAs and the sequence similarity between miRNA family members, new and different methods are needed to detect and quantify their expression. Additionally, it is desirable to analyze the expression levels of miRNA precursors. For example, miRNA precursor levels can provide an indirect method of analyzing the expression levels of mature miRNAs. Studying the differential expression of different miRNA precursors as compared to the mature miRNA is itself of interest. For example, certain disease processes may interfere with different steps during the processing of miRNA precursors.
Therefore, a need exists for a high throughput method that allows for the simultaneous analysis of miRNA precursor molecules and that provides for the analysis of miRNA expression when only small amounts of starting material are available.